Hypoxic HK2 Gene Regulation Involves Multiple Distinct Cis‐Acting Elements That Differ In Their Hypoxia‐Inducible Factor‐α (HIFα) Isoform Selectivity

Abstract

As the only effective energy substrate in the absence of O2, glucose (Glc) is central to all cellular strategies for hypoxic adaptation. Obligatory reliance on Glc also explains associated induction of glycolytic enzymes such as hexokinases which catalyze the first committed step of Glc metabolism. HIF1α contributes to HK2 expression in cancer cells, but the specific cis‐acting sequences are undefined and corresponding effects in nontransformed cells are unexplored. We therefore used chimeric reporter genes to delineate hypoxia‐responsive cis‐acting HK2 promoter regions in nontransformed NRK‐52E cells.Major hypoxic control of HK2 expression mapped to a phylogenetically conserved tandem pair of canonical HIF‐binding motifs just 5' to the 157 bp basal promoter region. Targeted mutagenesis of either motif abrogated hypoxia‐responsiveness, as did their spatial separation by a half (+5 bp) ‐ but not a full (+10 bp) ‐ helical turn. Similar results with hypoxia‐mimetics (e.g. CoCl2) and stabilized mutants of HIF1α ‐ but not HIF2α ‐ suggest HIF1α selectivity. Independent hypoxic enhancer activity responsive to both HIF1α and HIF2α ‐ but not other known HK2 transcriptional regulators ‐ was also observed within 90 bp of the transcription start site. These results suggest complex hypoxic gene regulation involving both combinatorial interactions and cis‐acting elements with differing HIFα isoform specificities.